Phytosanitary problems detected in truffle cultivation in Spain . A review

Aim of study: In black truffle plantations, many factors are driving the emergence of new pests and diseases which in forestry areas go unnoticed. Usually, the incidence of most of them is low. Nevertheless, in specific cases, some of them are capable of causing irreversible damage that could endanger the harvest or even tree survival. Area of study: This paper presents an in-depth study of the pests and diseases more frequently associated with truffle plantations in the region of Aragón (Spain). Damages have been arranged into the main production stages: nursery, plantations and fruiting bodies. Material and methods: The data used in this work come from the technical enquiries from truffle growers to researchers and technical staff, as well as from field visits undertaken by those work teams. Sampling methodology has been carried out following standard procedures. Insects were collected with the traps commonly used in entomology works. Main result: More than 50 damages have been described in this paper. Some of them are capable of weakening the plants and other can even kill them. Mycorrhizal competitor fungi have also been considered in this paper. These organisms do not cause real phytosanitary problems, but they can lead to important economic losses. Research highlights: Researches, truffle hunters and managers must be alert in the face of the possible occurrence of potentially dangerous organisms. The final aim: being able to take action in an efficient way in the case of a pest outbreak.

Nowadays 10,000 ha are estimated to be used for truffle culture, of which approximately 6,000 ha are in the region of Aragón (North East Spain).Annual growth is estimated at 500 ha (Reyna et al., 2013).Due to the high profitability of these plantations and to the support of the public authorities, Spain is becoming a world leader in this activity.But this is not the only marketable truffle in Spain.Other truffles, as Tuber aestivum Vittad., or Tuber brumale Vittad.are also collected and traded (De Román and Boa, 2004).However, their lower cost and the lack of knowledge about their culture are directing all efforts of truffle growers and researchers towards an increase of T. melanosporum production.
As the trend to monoculture grows and plantation densities increase, the quantity of pests associated with truffle plantations keeps rising.In fact, insects that lived in balance in the natural environment have turned to pests in recent years.
This paper presents a comprehensive study of pests and diseases detected in truffle plantations in Spain.

Material and methods
Study area involves truffle plantations and forest, mainly in the region of Aragón.This is a spanish region with optimal eco-climatic conditions for black truffle development.This fact is reflected in the total number of truffle plantations and in the high yields that are obtained from this activity.
Sampling methodology has been carried out following standard procedures.Insects were collected with the traps commonly used in entomology works.Traps include Malaise trap (Townes, 1972), handmade budget sleeves and yellow sticky cards (CROMATOL ® Halper Industria química, S.L. Pontevedra, Spain).Visual inspection of damaged trees has been also used to identify the pathogenic causal agent.
After trapping, samples were carried to the laboratory.Mainly, the works carried out by De Liñán (1998), Fraval (1998), Agrios (1998) and a database from the University of California (http://www.ipm.ucdavis.edu/) were used to identify all the insects appearing in plantations, forests and nurseries.
The results of this work are arranged in the three main stages of truffle culture: nursery, plantation and fruiting bodies.Mycorrhizal competitor fungi have also been considered in this paper.These organisms do not cause real phytosanitary problems, but they can lead to important economic losses.Ectomycorrhizal types were identified by anato-morphological features according to Agerer (2006) and Agerer andRambold (2004-2013).

Pests associated with nurseries
As for plant production in nurseries, pathogenic fungi, competitor fungi with T. melanosporum mycorrhizas and arthropods have been detected (Table 1).
The presence of powdery mildew (Microsphaera alphitoides Griffon & Maublanc) (Suppl.Fig. S1 [pdf online]) is common in plants established in the field.Furthermore, it also appears quite frequently on oaks [Quercus robur L., Quercus petraea (Matts.)Liebl., Q. faginea, Q. humilis, etc.] during the nursery phase.Its proliferation is normally related to excess moisture, and it is treated with sulfur or its derivatives (Zambonelli, 1993).This procedure, although effective, must be carried out with the necessary precautions when working with inoculated plants, as this antifungal treatment can endanger the correct mycorrhization process.Days when high temperature occurs should be avoided, since a phytotoxicity effect can be produced.
Other fungi in nurseries are able to compete with T. melanosporum mycorrhizae.This is the case of mycorrhizal fungus Sphaerosporella brunnea (Alb.& Schwein.)Svrc ˘ek y Kubic ˘ka (Suppl.Fig. S2 [pdf online]).This ascomycete is a pioneer fungus able to form mycorrhizae faster than T. melanosporum.Thus, it can displace the latter from its host plant as it infects root tips.S. brunnea limits the proper plant mycorrhization, and therefore its quality, sometimes even causing economic losses (Sánchez, 2012).It is difficult to eradicate when present in greenhouses because its control is achieved by managing moisture and temperature conditions (Sánchez et al., 2014).Nevertheless, this species shows low competition under field conditions and it does not cause problems in plantations (Hall et al., 2007).
Other ectomycorrhizal fungi can be found in nurseries.This is the case of Tuber albidum Complex (Suppl.Fig. S3 [pdf online]), and Trichophaea woolopheia (Cooke & W. Philips) Boud.(Suppl.Fig. S4 [pdf online]).Their presence is usually due to the use of poorly disinfected soil from forestry areas when preparing substrate (Garcia-Barreda, 2011).Other species of the genus Tuber, such as T. brumale, are sometimes detected.This situation is due to a mistake in the inoculum selection, although this is not common.
Different arthropods are relatively common in mycorrhizal plant production nurseries, although damages are usually not relevant and they do not weaken the plants excessively.They include the Coleoptera Otiorhynchus sulcatus Fabricius (Casteels and De Clercq, 1988)  Due to their large size and high caloric content in lipids and carbohydrates, acorns are a good source of food.Therefore it is easy to find in them certain insects such as the weevil Curculio elephas Gyllenhal (Bonal et al., 2007) et al., 2008).These insects can cause an important decrease in acorn germination (Soria et al., 1996).

Galls
Attacks of mice (Apodemus sylvaticus L.) and voles (Microtus sp.Schrank ) have been rarely detected in seedbeds.A wrong management in nurseries can lead to abiotic damages caused by frosts, water stress or chlorosis due to a nutrient deficiency in the substrate.On the other hand, an inadequate nursery location that leads to achieving a poor seedling hardening may endanger plant survival in the field (Mollá et al., 2003).

Pests associated with truffle plantations
In recent years, different diseases have appeared in truffle plantations (Table 1).Some of them are capa-ble of weakening the plants and other can even kill them.

Damages identified in the crown
The scale insects Kermes vermilio Planch.and Kermes ilicis L. (Suppl.Fig. S9 [pdf online]) are capable of causing significant damages in branches and trunks.Due to their high incidence, these Homoptera are a real problem in Spanish plantations.Like all sucking insects, direct damages are associated with sap suction.These scale insects weaken the attacked tree, causing a progressive yellowing in leaves and terminal branches, and thus reducing the amount of green shoots (Malumphy, 2008;Spodek and Ben-Dov, 2012).Damages caused by a massive attack of these insects usually do not endanger plant survival, but they can   (Malumphy, 2008;Martín Bernal et al., 2002).Their mobility is not high, so wind and birds must have an important role in the spreading of this insect (García Marí et al., 1989).
Targionia vitis Sign.(Suppl.Fig. S10 [pdf online]) is another scale insect which shows a lower incidence.Nonetheless, it is still capable of inducing a weakening in truffle trees.
The Buprestidae Coroebus florentinus Herbst (Suppl.Fig. S11 [pdf online]) bores galleries that spiral round the branch inner bark and interrupt sap flow, which causes it to dry out.It also causes an imbalance between aerial and root parts.
Cossus cossus L. and Zeuzera pyrina L. (Fig. 1) (Cossidae) can feed on young shoots.If an attack is conducted on the second or third year after plantation, the tree may die.
The proliferation in recent years of some tumor malformations (Suppl.Fig. S12 [pdf online]) appearing in branches and trunk in evergreen oaks (Q.ilex) deserves a special mention.Symptoms begin with a chlorotic pattern in younger leaves, followed by defoliation.Oversized malformations develop wherever there are open wounds in the bark.Over time, there is a general weakening of the tree and in some cases it results in death.Recent studies show that a Candidatus Phytoplasma is the cause of this disease (Martín et al., 2012).
Within the defoliating complex there is a great number of phytophagous insects.Most of them are Lepidoptera.Three of the most voracious insects, although rare in truffle plantations, are Lymantria dispar L. (Suppl.Fig. S13 [pdf online]), Tortrix viridiana L. (Suppl.Fig. S14 [pdf online]) and Euproctis chrysor-rhoea L. (Suppl.Fig. S15 [pdf online]).L. dispar and E. chrysorrhoea are two polyphagous species (Frago et al., 2010;Schultz and Lechowicz, 1986), while T. viridiana is monophagous on Quercus but polyphagous within the taxonomic limits of this genus (Du Merle, 1998).In young trees belonging to Q. ilex species, a complete defoliation can cause the death of the tree.
There are many other species typically present in plantation tree canopies.However, they keep low population levels and they only are capable of causing real damages when a population explosion occurs.This is the case of Chrysomelidae Lachnaia hirta Fabricius, Agelastica sp.L. and Labidostomis spp.Dejean (Suppl.Fig. S16 [pdf online]) and the weevil beetle Polydrusus setifrons Jacquelin du Val.
The presence of Eriophyid mites in oaks is very common, mainly Aceria ilicis Canestrini.This mite causes erinea in leaves.The affected leaves initially turn into a yellowish colour that later evolves into a dark brown.
The presence of aphids (Suppl.Fig. S17 [pdf online]) in truffle plantations is frequent.They may cause a weakening of the plant or a leaf mass decrease if the tree is young.These insects are frequently accompanied by sooty mold.
Phylloxera quercus usually appears in Q. faginea, Q. robur and Q. ilex.Its symptoms consist of yellow spots on leaves coinciding with suction points.These spots, over time, turn to brown and can even cause leaf drop.
Galls (Suppl.Fig. S18 [pdf online]) are defined as an evolutionary response that many vegetables show to the action of different specific organisms.Many species are capable of inducing these structures, such as fungus, bacteria or arthropods.This latter group is the largest, and it includes mainly species of the Cynipidae family.More than 140 Cynipidae species are gall formation inducers.Among them, approximately 70% are associated to trees belonging to the Quercus genus (Nieves-Aldrey, 1998).Galls damages are not usually serious, except in those cases in which an imbalance in the ecosystem prevents hyperparasites from controlling the pest population efficiently.Some of the most frequent species in the truffle plantations of Aragón are: Neuroterus quercusbaccarum L., Neuroterus numismalis Fourcroy, Andricus quercusramuli L., Andricus quercustozae Bosc, Plagiotrochus quercusilicis Fabricius and Dryomyia lichtensteinii F. Löw (Suppl.Fig. S19 [pdf online]).(Perez-Izquierdo and Pulido, 2013).It also produces in species of the genus Quercus, small longitudinal lesions of a few centimetres on the trunk bark which produce exudates, especially during spring and autumn.
Botryospheria stevensii Shoemaker (anamorph Diplodia mutila Fr.Mont.) (Suppl.Fig. S22 [pdf online]) is a fungus that causes death of twigs and branches.First symptoms consist of groups of chlorotic leaves.After this, leaf colour becomes brown.In the branches, the bark becomes necrotic, and longitudinal wounds appear.Below the diseased bark, wood colour degenerates into a reddish brown.Attacked trees show twigs with dry tips and branch dieback.

Damages identified in the tree root system
Several arthropod species are capable of causing lesions in roots.Among them, the following must be highlighted: the Curculionidae Otiorhynchus sulcatus, the Cerambycidae Vesperus sp.Dejean, different Melolonthidae of the genus Melolontha Fabricius (Suppl.Fig. S23 [pdf online]) and Anoxia Laporte, and Elateridae known as "click beetle".All these insects can cause severe injuries when the plant is young, even death.Attacks take place during larval stage, in which the individuals are equipped with very powerful jaws.
However, not only arthropods can cause damages to the root system.Ascomycete fungus Rosellinia necatrix Berl ex.Prill and Basidiomycete Armillaria mellea (Vahl) P. Kumm (Suppl.Fig. S24 [pdf online]) cause wood decay.The origin of this problem usually resides in a preceding woody crop.It is associated to water excess, generally due to a shallow water table (Ono, 1970;Ayres and Boddy, 1986).
An extremely serious problem found in the evergreen oak savannas (dehesas) from Andalucía and Extremadura (Spain) (Sánchez et al., 2002) but still not detected in truffle plantations in Aragón is the Oomycete Phytophthora cinnamomi De Bary.It occurs when the plant has suffered water stress in the presence of this organism.
P. cinnamomi and B. quercina have both been related to the oak decline in Spain (Soria et al., 1997).This is a very serious disease that is widely spread in Spanish holm dehesas.Fortunately, it has not yet become a problem in truffle plantations.
Another species of this genus, P. ramorum, has proven to be infective under controlled conditions on Quercus (Moralejo et al., 2009).Nevertheless it has not yet been demonstrated its presence on Quercus in Spain.Care in handling plant nursery is essential to prevent its occurrence in plantations.
Due to the uncontrolled proliferation of wild boars (Sus scrofa L.) in Spain, problems generated by this species have increased in recent years, mainly in wild truffle stands.This fact forces truffle growers to fence their fields when establishing the plantation.These mammals dig in the ground leaving the roots exposed, causing a great loss of root mass.Voles (Suppl.Fig. S25 [pdf online]) and mice also cause root damage.These rodents cause wounds in the root neck, and may lead to tree death.However, they play an important role in the dispersion of spores (Frank et al., 2008).
Otherwise, ectomycorrhizal species Trichophaea woolhopeia (Cooke & W. Phillips) Boud., Pisolithus arhizus (Scop.)Rauschert (Suppl.(Suppl.Fig. S28 [pdf online]) have been considered in this research work.These fungi are the most common in truffle plantations and belong to the mycorrhizal courtship of T. melanosporum.However, the role of these species in black truffle fruiting is still unknown nowadays.Several works (De Román and De Miguel, 2001;Reyna et al., 2006;Rubini et al., 2011;De Miguel et al., 2014) report the frequent identification of these species in truffle plantations, but they still have not been related to the life cycle of T. melanosporum.

Pests associated with fruiting bodies
To complement this study, problems in fruiting bodies have been identified (Table 1).In this sense, insects and other animals have a dual role.On one hand, they behave as predators that feed on truffles and, on the other hand, they spread the spores.
Its presence in truffle fruiting bodies is proven since the early twentieth century.Pradel in 1914 and Arzone in 1971, exposed the existing problems with these arthropods, but damages caused by these species are of increasing concern among Spanish truffle growers since the intensification of truffle orchards.On one hand, galleries of larvae cause product depreciation as consumers perceive that larvae and their faeces may be present.And, on the other hand, larva feeding causes a decrease in truffle weight (Barriuso et al., 2012).
H. tuberivora (Fig. 2) has been detected both in T. melanosporum and T. aestivum carpophores.Their larvae, and therefore their galleries, are generally smaller in size.However, there are usually a high nu mber of larvae feeding on a single truffle.This can reduce up to 40% the fresh weight of the fruiting body.Quality loss is unavoidable because a fast putrefaction and enzymatic degradation occurs throughout the gleba (Rivera et al., 2010).
L. cinnamomea (Suppl.Fig. S29 [pdf online]), is a species strictly linked to Tuber species mainly T. melanosporum (Pacioni et al., 1991).The proliferation of this beetle in truffle plantations is very high, raising concern among truffle growers and managers.The damages are severe, although putrefaction of the specimens is slower than in the previous case.
There is no effective treatment for these pests.Only cultural actions are carried out to reduce the populations of these insects.A superficial tillage (30-40 cm) to prevent truffle development in the first centimeters and the collection in the plantations of truffles from species different to T. melanosporum are the two re-commended measures to prevent the spread of these insects.
Other insects have been detected in carpophores, such as Pleurophorus sp.Mulsant (Suppl.Fig. S31 [pdf online]) or the myriapod of the Class Diplopoda Ommatoiulus sabulosus L. (Suppl.Fig. S32 [pdf online]).These are usually detritivorous and coprophagous species, so their presence in fruiting bodies is due to the prior presence of galleries from other parasites.

Conclusions
The intensification of truffle plantations in Spain has led to the emergence of a large number of pests and diseases associated with this crop.Nevertheless, few of them can be considered dangerous or capable of causing irreversible damage to plantations.Among all of them Kermes sp. and the tumor malformations recently identified as a Candidatus Phytoplasma are highlighted.The ectomycorrhizal fungus S. brunnea is a problem in nurseries that produce mycorrhizal plants because it lowers T. melanosporum colonization and keeps seedlings from meeting quality standards.
Choosing an inappropriate acorn provenance and the use of species not adapted to the plantation area are causing in some cases the plant inadequacy, which leads to poor growth and, in extreme cases, tree death (Andivia et al., 2012;Del Campo et al., 2010).
The role of research in phytosanitary issues is clear: potentially dangerous organisms must be identif ied including their biology, etiology and control measures.The final aim: being able to take action in an efficient way in the case of a pest outbreak.Truffle culture is an organic crop (Reyna, 2012).To try and follow this philosophy, any action performed on a truffle plantation to eliminate harmful organisms should be conducted according to the principles of organic farming.Whenever possible, agronomic, biological, and mechanical methods must be applied, as opposed to the use of chemical substances.The search for balance in the ecosystem is an objective to be achieved, trying to promote the existence of native auxiliary fauna and ensuring a proper plant development.
Erinea in leaves.The affected leaves initially turn into a yellowish colour that later evolves into dark brown Mines in the upper surface of leaves Yellow and brown spotting of leaves.General browning of the foliage and premature leaf fall Presence of aphids in shoots, frequently accompanied by sooty mould Holes in the acorns.Compact droppings inside acorns Holes in the acorns.Granulated droppings inside acorns Extraction of part of the potting mix from the seedling containers.Absence of acorns in containers Presence of females on the twigs and branches Presence on stems and branches, especially under the bark Yellowish brown branches, evolving later into a reddish tone.Presence of galleries that spiral around the branch growth of young plants and may kill tree seedlings Rejection of the batch depending on the method used for seedling quality evaluation Rejection of the batch Rejection of the batch depending on the method used for seedling quality evaluation Rejection of the batch Weakness and subsequent death of affected plants Weakness and malformations in affected plants.Decrease of the seedling economic value due to poor appearance Seedlings usually do not suffer excessively with this pest.Decrease of the seedling economic value due to poor appearance Weakening of the seedling.Decrease of the seedling economic value due to poor appearance Normally of no importance but persistent attacks on young trees reduce plant vigour Weakening of the plant Economic losses due to germination failure Economic losses due to germination failure Economic losses due to germination failure Weakening of the plant even death in case of persistent attacks Weakening of the plant Imbalance between aerial and root parts due to galleries, which interrupt the sap flow.Weakening of the plant.Brittle branches Presence of sawdust and/or droppings.Presence of larvae and galleries under the bark Presence of newly hatched larvae in young twigs, to be found later in larger branches or trunks Insect egg depositions on vertical or overhanging surfaces, usually trunks, and never on the floor Symptoms visible in the tree crowns including complete defoliation when larvae are found in large numbers Defoliation.Gregarious and highly urticating larvae, which spin a very visible web around them Presence of larvae feeding on sprouts Presence of adults in the tree crown They feed on parenchyma, leaving the leaf veins intact Erinea in leaves.The affected leaves initially turn into a yellowish colour that later evolves into a dark brown Presence of aphids in shoots, frequently accompanied by sooty mould Yellow and brown spotting of leaves.General browning of the foliage and premature leaf fall Presence of galls, malformations or thickenings with different shapes Exudates on stems, branches, twigs or acorns Reddish-brown coloration of the plant.Presence of larvae in affected plants Presence of larvae in root system.Reddish-brown coloration of the plant Torn and chewed young plants.Soil profile disturbance Vigour decline, yellowing foliage, reduced leaf size and number.Mycelium in roots Smaller yellowing leaves.Short grouped twigs Exudates on stems, branches, twigs or acorns and root parts due to galleries, which interrupt sap flow.Weakening of the plant.Brittle branches Imbalance between aerial and root parts due to galleries, which interrupt sap flow.Weakening of the plant.Brittle branches Severe defoliation.Weakening of the plant, leading sometimes to disease and/or death Weakening of the plant.Reduction of acorn production Defoliation caused by larvae, which eat the buds and young leaves.Weakening of the plant Weakening of the plant.Malformations Defoliation, decreased flowering Weakening of the plant.Reduction of plant vigour Slightly deformed leaves.Gall-like thickenings and deformations in inflorescenses Weakening of the plant Normally of no importance in old plantations, but persistent attacks on young trees reduce plant vigour Malformations and alterations in twigs, stems, leaves and catkins Disturbance of the vegetative growth process Death of the plant Death of the plant Death of roots, decreasing of truffle production Weakening and even death of the plant Falling of the anomalous leaves Disturbance of the vegetative growth process culio glandium Marsham or the Lepidoptera Cydia fagiglandana Zeller (Torres Villa plant, even death if tumors affect the trunk Colonization of root tips preventing Tuber melanosporum presence in them Colonization of root tips preventing Tuber melanosporum presence in them Colonization of root tips preventing Tuber melanosporum presence in them Production of a non-desirable truffle in the plantations Truffle devaluation, decrease of truffle weight, rot of fruiting bodies Truffle devaluation, decrease of truffle weight, rot of fruiting bodies Truffle devaluation, decrease of truffle weight, rot of fruiting bodies Truffle devaluation, decrease of truffle weight, rot of fruiting bodies Spreading of microorganisms that cause truffle rot Spreading of microorganisms that cause truffle rot induce a total foliage loss

Figure 2 .
Figure 2. Digestive tract of the truffle fly Helomyza tuberivora containing black truffle spores.

Table 1 .
(Suppl.Fig.S8[pdf online]), Cur-Diseases or competitors present in each truffle cultivation stage.Their identification, incidence, danger risk and impact when the problem occurs are shown for each pest

Table 1 (
cont.).Diseases or competitors present in each truffle cultivation stage.Their identification, incidence, danger risk and impact when the problem occurs are shown for each pest

Table 1 (
Phytosanitary problems in truffle cultivation.A review 311 cont.).Diseases or competitors present in each truffle cultivation stage.Their identification, incidence, danger risk and impact when the problem occurs are shown for each pest